Obesity-related diseases, including type 2 diabetes and cardiovascular disease, have reached epidemic proportions in industrialized nations, and dietary interventions for their prevention are ...therefore important. Resistant starches (RS) improve insulin sensitivity in clinical trials, but the mechanisms underlying this health benefit remain poorly understood. Because RS fermentation by the gut microbiota results in the formation of physiologically active metabolites, we chose to specifically determine the role of the gut microbiota in mediating the metabolic benefits of RS. To achieve this goal, we determined the effects of RS when added to a Western diet on host metabolism in mice with and without a microbiota.
RS feeding of conventionalized mice improved insulin sensitivity and redressed some of the Western diet-induced changes in microbiome composition. However, parallel experiments in germ-free littermates revealed that RS-mediated improvements in insulin levels also occurred in the absence of a microbiota. RS reduced gene expression of adipose tissue macrophage markers and altered cecal concentrations of several bile acids in both germ-free and conventionalized mice; these effects were strongly correlated with the metabolic benefits, providing a potential microbiota-independent mechanism to explain the physiological effects of RS.
This study demonstrated that some metabolic benefits exerted by dietary RS, especially improvements in insulin levels, occur independently of the microbiota and could involve alterations in the bile acid cycle and adipose tissue immune modulation. This work also sets a precedent for future mechanistic studies aimed at establishing the causative role of the gut microbiota in mediating the benefits of bioactive compounds and functional foods.
Many enteric pathogens, including Salmonella and enteropathogenic and enterohemorrhagic Escherichia coli, express adhesins that recognize and bind to carbohydrate moieties expressed on epithelial ...cells. An attractive strategy for inhibiting bacterial adherence employs molecules that mimic these epithelial binding sites. Prebiotic oligosaccharides are non-digestible, fermentable fibres capable of modulating the gut microbiota. Moreover, they may act as molecular decoys that competitively inhibit adherence of pathogens to host cells. In particular, galactooligosaccharides (GOS) and other prebiotic fibres have been shown to inhibit pathogen adherence to epithelial cells in vitro. In the present study, we determined the ability of prophylactic GOS administration to reduce enteric pathogen adherence both in vitro and in vivo as well as protect against intestinal inflammation. GOS supplementation significantly reduced the adherence of the epithelial-adherent murine bacterial pathogen Citrobacter rodentium in a dose-dependent manner to the surface of epithelial cells in vitro. A 1- to 2-log reduction in bacterial adherence was observed at the lowest and highest doses tested, respectively. However, mouse studies revealed that treatment with GOS neither reduced the adherence of C. rodentium to the distal colon nor decreased its dissemination to systemic organs. Despite the absence of adherence inhibition, colonic disease scores for GOS-treated, C. rodentium-infected mice were significantly lower than those of untreated C. rodentium-infected animals (P=0.028). Together, these data suggest that GOS has a direct protective effect in ameliorating disease severity following C. rodentium infection through an anti-adherence-independent mechanism.
Cronobacter sakazakii is now recognized as an opportunistic pathogen and has been implicated in rare but severe cases of necrotizing enterocolitis, meningitis, and sepsis in neonates. The first step ...in bacterial pathogenesis requires that the organism adheres to host cells surfaces; therefore, agents that inhibit adherence might be useful for preventing infections. Lactoferrin, an iron binding protein found in milk, has been shown to inhibit bacterial adherence by direct interaction and disruption of bacterial surfaces. Therefore, the goal of this research was to assess the ability of two different types of bovine lactoferrin, alone and in combination with a 1:1 blend of galactooligosaccharides and polydextrose, to inhibit adherence of C. sakazakii to a HEp-2 human cell line. Results showed that the adherence of C. sakazakii was significantly reduced at a minimum lactoferrin concentration of 10 mg/ml. However, in combination with the oligosaccharide blend, no synergistic effect was observed in adherence inhibition. These results suggest that lactoferrin might interact with C. sakazakii and directly inhibit adhesion to tissue culture cells.
Some oligosaccharides are known to act as molecular decoys by inhibiting pathogen adherence to epithelial cells. The present study was aimed at analyzing whether chitooligosaccharides (CHOS), that ...is, oligomers of d-glucosamine and N-acetyl-d-glucosamine, have such antiadherence activity. CHOS of varied degree of polymerization (DP) and fraction of acetylation (FA) were produced. Adherence of enteropathogenic Escherichia coli (EPEC) to the surface of a human HEp-2 cell line was determined in the absence or presence of the various CHOS fractions. Adherence was assessed by microscopic counting and image analysis of bacterial clusters and cells. The results showed that all CHOS fractions inhibited adherence of EPEC to HEp-2 cells. Hydrolysates with lower FA were more effective at reducing adherence. This effect is greater than that obtained with other oligosaccharides, such as galactooligosaccharides, applied at the same concentrations.
Dysfunction in the neurovascular unit (NVU) is a key component in the progressive deterioration of Alzheimer’s disease (AD) and is critical in vascular dementia. Recent studies have shown that ...inflammation plays early and perhaps causal roles in the pathogenesis of AD related to NVU damage, possibly in part by overactivating the aspartic acid protease activity of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), which until now has almost solely been studied in the context of the β-amyloid cascade. In this study, we analyzed the relationship of BACE1 with astrocytes and blood vessels in human brains with sporadic and familial dementia Autosomal dominant cerebral arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), sporadic Alzheimer’s disease (SAD), and familial Alzheimer’s disease (FAD) and how BACE1 inhibition affects astrocytes and endothelial cells under conditions of glutamate toxicity. Our results show increased BACE1, PHF (Paired helical filaments)-tau and GFAP (Glial Fibrillary Acid Protein) immunoreactivity (IR) in the CA1 hippocampal regions of FAD and SAD brains. Furthermore, BACE1 immunoprecipitated with GFAP in tissue samples from all study cases, but their immunofluorescence close to (10 μm
3
) or overlapping blood vessels was only increased in FAD and SAD brains, and PHF-tau was present around the vessels mainly in FAD brains. Interestingly, the increased BACE1 levels were associated with reactive astrocytes, characterized by morphological changes and upregulation of GFAP under pathological and stressful conditions, and endothelial disruption by glutamate excitotoxicity, and these effects were reversed by BACE1 inhibition; further, BACE1-inhibited astrocytes protected endothelial cell integrity by preserving zonula occludens-1 (ZO-1) distribution and decreasing the expression of inflammatory markers. Taken together, these findings suggest that BACE1 dysregulation in astrocytes may have a role in the alterations in NVU integrity implicated in neurodegeneration.
The development of sustainable extraction methods to obtain natural products constitutes a challenge for the food industry. The aim of this work was to compare yield, separation efficiency, chemical ...composition, and physicochemical properties of the mucilage extracted from fresh cladodes (FNM) and mucilage extracted from dehydrated cladodes (DNM) of O. ficus indica. Suspensions of fresh and dehydrated cladodes (4% w/w) were prepared for mucilage extraction by using a mechanical separation process. Subsequently, the separated mucilage was precipitated with ethyl alcohol (1:2 v/v) then, yield and separation efficiency were determined. The mucilage was characterized by measuring Z potential, viscosity, color, and texture attributes. Additionally, chemical proximate analysis, scanning electron microscopy, and thermogravimetric analysis (TGA) were conducted. No significant differences (p < 0.05) were detected in the yield and separation efficiencies between samples. Nevertheless, the dehydration process of cladodes prior to mucilage extraction increased protein, ashes, nitrogen free extract, and calcium content. The viscosity was higher in DNM than in FNM. The TGA revealed a different thermal behavior between samples. In addition, the DNM showed lower L (darkness/lightness), cohesiveness, adhesiveness, and springiness values than those of FNM. These results support that differences found between the chemical and physicochemical properties of DNM and those of FNM will determine the applications of the mucilage obtained from the O. ficus indica cladodes in the food, pharmaceutical, and cosmetic industries.
Mesenchyme homeobox-2 (MEOX2)-mediated regulation of glioma-associated oncogene-1 (GLI1) has been associated with poor overall survival, conferring chemoresistance in lung cancer. However, the role ...of MEOX2/GLI1 in resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs)-based therapy remains unexplored in human lung cancer.
Functional assays using genetic silencing strategy by short hairpin RNAs, as well as cytotoxic (tetrazolium dye MTT) and clonogenic assays, were performed to evaluate MEOX2/GLI1-induced malignancy capacity in lung cancer cells. Further analysis performed includes western blot, qPCR and ChIP-qPCR assays to identify whether MEOX2/GLI1 promote EGFR/AKT/ERK activation, as well as EGFR overexpression through epigenetic mechanisms. Finally, preclinical tumour progression in vivo and progression-free disease interval analyses in patients treated with EGFR-TKI were included.
Overexpressed MEOX2/GLI1 in both EGFR wild-type and EGFR/KRAS-mutated lung cancer cells were detected and involved in the activation/expression of EGFR/AKT/ERK biomarkers. In addition, MEOX2/GLI1 was shown to be involved in the increased proliferation of tumour cells and resistance capacity to cisplatin, EGFR-TKIs (erlotinib and AZD9291 ‘osimertinib’), AZD8542-SMO, and AZD6244-MEKK1/2. In addition, we identified that MEOX2/GLI1 promote lung tumour cells progression in vivo and are clinically associated with poorer progression-free disease intervals. Finally, both MEOX2 and GLI1 were detected to be epigenetically involved in EGFR expression by reducing both repressive markers polycomb-EZH2 and histone H3K27me3, but, particularly, increasing an activated histone profile H3K27Ac/H3K4me3 at EGFR-gene enhancer-promoter sequences that probably representing a novel EGFR-TKI-based therapy resistance mechanism.
MEOX2/GLI1 promote resistance to cisplatin and EGFR-TKI-based therapy in lung cancer cells, modulating EGFR/AKT/ERK signalling pathway activation, as well as inducing an aberrant epigenetic modulation of the EGFR-gene expression in human lung cancer.
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•Resistance to EGFR-TKI based treatment occurs through MEOX2/GLI1 transcription factors in lung cancer cells.•Overexpressed MEOX2/GLI1 promotes cisplatin resistance and lung tumour cells proliferation.•MEOX2/GLI promotes in vitro resistance to EGFR-TKI therapy by EGFR-pathway over-activation.•Overexpressed MEOX2/GLI1 increase in vivo lung tumour cells progression.•MEOX2/GLI1 modulate genetically and epigenetically to EGFR-gene expression promoting lung malignant progression.
Nailfold videocapillaroscopy (VCP) allows non-invasive assessment of the microcirculation. Adequate training in this field is relevant for rheumatologists. There is increasing evidence of the ...reliability of VCP findings among different readers. Objective: To evaluate inter- and intra-reader agreement of rheumatologists to identify normal images and systemic sclerosis (SSc) patterns on VCP (“early,” “active,” and “late” proposed by Cutolo et al.). Thirteen rheumatologists with different experience in nailfold VCP received training to standardize reading criteria. They rated 60 VCP images from healthy and SSc patients at baseline and 4 weeks later, using an electronic platform. The reading of an expert was considered the gold standard. Data were analyzed using Cohen’s kappa for concordance and Student’s
t
test and ANOVA to compare kappa means for inter-reader, intra-reader, and inter-pattern readings. Mean inter-reader and intra-reader kappa were 0.45 and 0.49, respectively, (moderate agreement). Kappa scores were higher among experienced vs inexperienced readers (inter-reader kappa 0.58 vs 0.34,
p
= 0.001, intra-reader kappa 0.65 vs 0.37,
p
= 0.01). Agreement was substantial (kappa = 0.61) for the identification of normal vs abnormal images and higher for the identification of active (0.48,
p
= 0.009) and late SSc patterns (0.56,
p
= 0.008) than for the early SSc pattern (0.35,
p
= 0.003). There is moderate agreement among rheumatologists for the identification of SSc videocapillaroscopy patterns (higher among experienced rheumatologists) and substantial agreement, regardless of previous experience in VCP, in the identification of normal and abnormal images. Agreement for the identification of active and late patterns is higher than for the early pattern.
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